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Genetic and functional studies of hereditary myopathy with lactic acidosis
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics. (Monica Holmberg)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Genetiska och funktionella studier av hereditär myopati med laktacidos (Swedish)
Abstract [en]

Hereditary myopathy with lactic acidosis (HML, OMIM#255125) is an autosomal recessive disorder which originates from Västerbotten and Ångermanland in the Northern part of Sweden. HML is characterized by severe exercise intolerance which manifests with tachycardia, dyspnea, muscle pain, cramps, elevated lactate and pyruvate levels, weakness and myoglobinuria. The symptoms arise from malfunction of the energy metabolism in skeletal muscles with defects in several important enzymes involved in the TCA cycle and the electron transport chain. All affected proteins contain iron-sulfur (Fe-S) clusters, which led to the suggestion that the disease was caused by malfunctions in either the transportation, assembly or processing of Fe-S clusters.

The aim of my thesis was to identify the disease causing gene of HML and to investigate the underlying disease-mechanisms. In paper I we identified a disease-critical region on chromosome 12; a region containing 16 genes. One of the genes coded for the Fe-S cluster assembly protein ISCU and an intronic base pair substitution (g.7044G>C) was identified in the last intron of this gene. The mutation gave rise to the insertion of intron sequence into the mRNA, leading to a protein containing 15 abberant amino acids and a premature stop. In paper II we investigated why a mutation in an evolutionary well conserved protein with a very important cellular role, which in addition is expressed in almost all tissues, gives rise to a muscle-restricted phenotype. Semi-quantitative RT-PCR analysis showed that the mutant transcript constituted almost 80% of total ISCU mRNA in muscle, while in both heart and liver the normal splice form was dominant. We could also show that, in mice, complete absence of Iscu protein was coupled with early embryonic death, further emphasizing the importance of the protein in all tissues. These data strongly suggested that tissue-specific splicing was the main mechanism responsible for the muscle-specific phenotype of HML. In paper III the splicing mechanisms that give rise to the mutant ISCU transcript was further investigated. We identified three proteins; PTBP1, IGF2BP1 and RBM39, that could bind to the region containing the mutation and could affect the splicing pattern of ISCU in an in vitro system. PTBP1 repressed the inclusion of the intronic sequence, while IGF2BP1 and RBM39 repressed the total ISCU mRNA level though the effect was more pronounced for the normal transcript. Moreover, IGF2BP1 and RBM39 were also able to reverse the effect of PTBP1. IGF2BP1, though not a splicing factor, had higher affinity for the mutant sequence. This suggested that the mutation enables IGF2BP1 binding, thereby preventing the PTBP1 induced repression seen in the normal case.

In conclusion, we have determined the genetic cause of HML, identifying a base pair substitution in the last intron of the ISCU gene that gives rise to abnormally spliced transcript. The muscle-specific phenotype was also analyzed and tissue-specific splicing was identified as the main disease-mechanism. Furthermore, nuclear factors with ability to affect the splicing pattern of the mutant ISCU gene were identified. This work has thoroughly investigated the fundamental disease mechanisms, thus providing deeper understanding for this hereditary myopathy.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2011. , 39 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1454
Keyword [en]
Hereditary myopathy with lactic acidosis, ISCU, intron mutation, mouse model, tissue-specific splicing
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
URN: urn:nbn:se:umu:diva-50592ISBN: 978-91-7459-308-2 (print)OAI: oai:DiVA.org:umu-50592DiVA: diva2:471543
Public defence
2012-01-27, Sal B, By 1D 9 tr, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-01-05 Created: 2011-12-14 Last updated: 2012-01-05Bibliographically approved
List of papers
1. Myopathy with lactic acidosis is linked to chromosome 12q23.3-24.11 and caused by an intron mutation in the ISCU gene resulting in a splicing defect
Open this publication in new window or tab >>Myopathy with lactic acidosis is linked to chromosome 12q23.3-24.11 and caused by an intron mutation in the ISCU gene resulting in a splicing defect
2008 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 17, no 11, 1666-1672 p.Article in journal (Refereed) Published
Abstract [en]

We describe the mapping and identification of the gene for hereditary myopathy with lactic acidosis (HML). HML is characterized by low physical performance, resulting in physical exertion that causes early exhaustion, dyspnoea and palpitations. Using an autosomal recessive mode of inheritance, we mapped the trait to chromosome 12q23.3-24.11, with a maximum lod score of 5.26. The 1.6-Mb disease-critical region contained one obvious candidate gene-ISCU-specifying a protein involved in iron-sulphur cluster assembly. IscU is produced in two isoforms; one cytosolic and one mitochondrial, coded for by different splice variants of the ISCU gene. Mutational analysis of all exon and intron sequences as well as 1000 bp of the promoter of the ISCU gene revealed one intron mutation that was specific for the disease haplotype. The mutation is located in a region with homology to the interferon-stimulated response element (ISRE), but we could not see any effect of the mutation on expression levels in vitro or in vivo. We did, however, observe a drastic difference in the splicing pattern between patients and controls. In controls the mRNA was, as expected, mainly in the mitochondrial form, while in the patients a larger mRNA transcript was predominant. Sequencing of the product revealed that the mutation activates cryptic splice sites in intron 5 resulting in aberrant mRNA containing 100 bp of the intron. To conclude, our data strongly suggest that an intron mutation in the ISCU gene, leading to incorrectly spliced mRNA, is the cause of myopathy with lactic acidosis in this family.

Keyword
succinate-dehydrogenase; hereditary myopathy; paroxysmal myoglobinuria; aconitase deficiency; cluster; identification; exercise
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-19316 (URN)10.1093/hmg/ddn057 (DOI)18296749 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2017-12-13Bibliographically approved
2. Tissue-specific splicing of ISCU results in a skeletal muscle phenotype in myopathy with lactic acidosis, while complete loss of ISCU results in early embryonic death in mice
Open this publication in new window or tab >>Tissue-specific splicing of ISCU results in a skeletal muscle phenotype in myopathy with lactic acidosis, while complete loss of ISCU results in early embryonic death in mice
2011 (English)In: Human Genetics, ISSN 0340-6717, E-ISSN 1432-1203, Vol. 129, no 4, 371-378 p.Article in journal (Refereed) Published
Abstract [en]

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulphur cluster assembly gene (ISCU) leading to incorporation of intron sequence into the mRNA. This results in a deficiency of Fe-S cluster proteins, affecting the TCA cycle and the respiratory chain. The proteins involved in the Fe-S machinery are evolutionary conserved and shown to be fundamental in all organisms examined. ISCU is expressed at high levels in numerous tissues in mammals, including high metabolic tissues like the heart, suggesting that a drastic mutation in the ISCU gene would be damaging to all energy-demanding organs. In spite of this, the symptoms in patients with HML are restricted to skeletal muscle, and it has been proposed that splicing events may contribute to the muscle specificity. In this study we confirm that a striking difference in the splicing pattern of mutant ISCU exists between different tissues. The highest level of incorrectly spliced ISCU mRNA was found in skeletal muscle, while the normal splice form predominated in patient heart. The splicing differences were also reflected at a functional level, where loss of Fe-S cluster carrying enzymes and accumulation of iron were present in muscle, but absent in other tissues. We also show that complete loss of ISCU in mice results in early embryonic death. The mice data confirm a fundamental role for ISCU in mammals and further support tissue-specific splicing as the major mechanism limiting the phenotype to skeletal muscle in HML.

Keyword
iron-sulfur proteins; succinate-dehydrogenase; paroxysmal myoglobinuria; deficiency; exercise; clusters; mutation; mitochondria; metabolism; maturation
National Category
Medical Genetics
Research subject
Medicine
Identifiers
urn:nbn:se:umu:diva-40798 (URN)10.1007/s00439-010-0931-3 (DOI)000289275200002 ()21165651 (PubMedID)
Available from: 2011-03-09 Created: 2011-03-09 Last updated: 2017-12-11Bibliographically approved
3. The defective splicing caused by the ISCU intron mutation in patients with myopathy with lactic acidosis is repressed by PTBP1 but can be de-repressed by IGF2BP1
Open this publication in new window or tab >>The defective splicing caused by the ISCU intron mutation in patients with myopathy with lactic acidosis is repressed by PTBP1 but can be de-repressed by IGF2BP1
2012 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 33, no 3, 467-470 p.Article in journal (Refereed) Published
Abstract [en]

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulfur cluster assembly gene ISCU which leads to the activation of cryptic splice sites and the retention of part of intron 4. This incorrect splicing is more pronounced in muscle than in other tissues, resulting in a muscle-specific phenotype. In this study, we identified five nuclear factors that interact with the sequence harboring the mutation and analyzed their effect on the splicing of the ISCU gene. The identification revealed three splicing factors, SFRS14, RBM39 and PTBP1, and two additional RNA binding factors, matrin 3 (MATR3) and IGF2BP1. IGF2BP1 showed a preference for the mutant sequence, whereas the other factors showed similar affinity for both sequences. PTBP1 was found to repress the defective splicing of ISCU, resulting in a drastic loss of mutant transcripts. In contrast, IGF2BP1 and RBM39 shifted the splicing ratio toward the incorrect splice form.

Keyword
ISCU;hereditary myopathy;alternative splicing;PTBP1
National Category
Basic Medicine
Identifiers
urn:nbn:se:umu:diva-50593 (URN)10.1002/humu.22002 (DOI)
Available from: 2012-01-02 Created: 2011-12-14 Last updated: 2017-12-08Bibliographically approved

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